Reagent for endotoxin assay and method for endotoxin assay using the same

ABSTRACT

This invention provides (1) a reagent for endotoxin assay which comprises aprotinin and a limulus amebocyte lysate reagent, (2) a kit for endotoxin assay which comprises the limulus amebocyte lysate reagent and a reagent containing aprotinin, (3) a method for assaying endotoxin in a sample using the limulus amebocyte lysate reagent in which aprotinin is added to the lysate reagent and/or the sample, (4) a method for assaying endotoxin in a serine protease-containing sample using the limulus amebocyte lysate reagent in which the sample is allowed to contact with an aprotinin-immobilized insoluble carrier in advance of endotoxin assay, (5) a carrier for pretreating a serine protease-containing sample on which aprotinin is immobilized, (6) a method for inhibiting factor G activation in which aprotinin is added to the limulus amebocyte lysate reagent and (7) a factor G activation inhibitor which comprises aprotinin as an active ingredient. 
     Endotoxin assay can be effected based on the factor C system reaction, without influences of factor G contained in the limulus amebocyte lysate reagent and/or serine proteases contained in samples.

This is a Continuation of application Ser. No. 08/202,177, filed on Feb.25, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates to a reagent for endotoxin assay, a kit forendotoxin assay, a method for endotoxin assay, a carrier for pretreatingsamples, a method for inhibiting activation of factor G and a factor Gactivation inhibitor, in which a limulus amebocyte lysate reagent isused.

BACKGROUND OF THE INVENTION

A method for assaying endotoxin (hereinafter referred to as "Et") usinga limulus amebocyte lysate (hereinafter simply referred to as "lysate")is well known as a limulus test. The assay comprising the reaction ofthe lysate is called a limulus reaction. A limulus test has so highsensitivity that is widely employed in pyrogen check of drugs and water,diagnostic use, and the like. The limulus test is based on coagulationof a lysate in the presence of a trace amount of endotoxin. The latestbiochemical study has elucidated the fact that the limulus reaction iscomposed of stepwise activation of several coagulation factors (J.Protein Chem., 5, 255-268 (1986)).

The limulus reaction is illustrated below with respect to a lysate fromTachypleus tridentatus by referring to FIG. 1. On endotoxin addition toa lysate, factor C (an endotoxin-sensitive factor; molecular weight:123,000) in the lysate is activated. The activated factor C limitedlyhydrolyzes factor B (molecular weight: 64,000) at a specific site toproduce activated factor B. The activated factor B activates proclottingenzyme (molecular weight: 54,000) to convert into clotting enzyme. Theclotting enzyme limitedly hydrolyzes coagulogen (coagulant protein;molecular weight: 19,723) at the specific sites in the loop crosslinkedby a disulfide linkage, i.e., intermediate between . . . Arg¹⁸ and Thr¹⁹. . . and intermediate between . . . Arg⁴⁶ and Gly⁴⁷ . . . to releasepeptide C (28 amino acid residues) represented by H-Thr¹⁹ . . . Arg⁴⁶-OH while converting the remaining part into coagulin gel. Thus, thelimulus reaction is composed of a series of reactions (cascade reactioncaused by endotoxin is hereinafter referred to as factor C systemreaction).

The above-mentioned cascade reaction of a lysate is induced by not onlyendotoxin but also a (1→3)-β-D-glucan (hereinafter simply referred to asa β-glucan). That is, factor G (a β-glucan-sensitive factor) in FIG. 1is activated by a β-glucan, the activated factor G converts proclottingenzyme into clotting enzyme, and clotting enzyme acts on coagulogen toproduce coagulin gel in the same manner of endotoxin as described above(cascade reaction caused by a β-glucan is hereinafter referred to asfactor G system reaction).

The clotting enzyme produced through the cascade reaction is alsocapable of hydrolyzing an amide linkage of a synthetic peptide substrateseparately added to the reaction system, such ast-butoxycarbonyl-leucyl-glycyl-arginine-paranitroanilide(Boc-Leu-Gly-Arg-pNA) to release paranitroaniline. Accordingly,endotoxin or the β-glucan can be quantitatively determined by measuringthe absorbance of the thus released paranitroaniline.

Since the generally used lysate contains components concerned in bothfactor C and factor G system reactions, its use for assaying endotoxinin a sample sometimes entails inaccurate result due to progress of thefactor G system reaction triggered by β-glucan possibly contained in thesample.

Thus, the limulus test was proved non-specific for the endotoxin assay,and a number of attempts have been made to develop a method forendotoxin-specific assay. For example, a method has been reported inwhich endotoxin-specific assay is effected by using a lysate fractionwhich contains only the factor C system reaction-related components(Obayashi T. et al., Clin. Chim. Acta, 149, 55-65 (1985)).

However, this method requires extremely complicated operations forpreparing a factor G-free system, including fractionation of a lysate byaffinity chromatography using an affinity carrier having immobilizedthereon dextran sulfate to eliminate the β-glucan-sensitive factor,i.e., factor G, and reconstruction of factor C, factor B and proclottingenzyme for use in endotoxin-specific assay.

On the other hand, it is known that all of these activated factorsinvolved in the limulus reaction (factor C and factor G systemreactions) are serine proteases, and the limulus test results in falsepositive in the presence of other serine proteases such as trypsin andthrombin which convert coagulogen into coagulin gel through its limitedhydrolysis or hydrolyze the above-mentioned synthetic substrate,nevertheless the presence or absence of Et and β-D-glucan (Harada T. etal., J. Med. Enzymol., 3, 43-60 (1978)). In consequence, it isimpossible so far to assay endotoxin in serine protease-containingsamples by limulus test.

SUMMARY OF THE INVENTION

A first object of the present invention is to specifically assayendotoxin in samples using a lysate reagent, by avoiding influence ofβ-glucan sensitive factor (factor G) contained in the lysate and thusbased on only the factor C system reaction.

A second object of the present invention is to accurately assayendotoxin in serine protease-containing samples using a lysate reagent,by excluding false positive reaction through specific inhibition of theactivity of serine proteases in the samples without inhibiting activityof the activated factors involved in the factor C system reaction in thelysate.

With the aim of attaining the first object described above, the presentinventors have examined substances capable of selectively inhibiting thefactor G system reaction, namely activation of factor G by β-glucanand/or activity of activated factor G, without inhibiting the factor Csystem reaction in the lysate. As a result, it was found that, amongvarious serine protease inhibitors, an appropriate amount of aprotinincan strongly inhibit the factor G system reaction without substantiallyinhibiting the factor C system reaction.

Also, with the aim of attaining the second object, the present inventorshave examined substances which can inhibit serine proteases in samplesselectively without inhibiting the factor C system reaction in thelysate. It was found as a result that serine proteases in samples can beinhibited selectively without inhibiting the factor C system reaction,by carrying out the limulus reaction in the presence of an appropriateamount of aprotinin as one of various serine protease inhibitors or byallowing samples to contact with an appropriate amount of aprotinin inadvance of the reaction.

We have examined various serine protease inhibitors such as α₁-antitrypsin, antithrombin III, α₂ -plasmin inhibitor, ovomucoidinhibitor, hirudine, gabexate mesylate and the like, in addition toaprotinin, and found that aprotinin alone can preferentially inhibit thefactor G system reaction in the limulus reaction, while other serineprotease inhibitors not only inactivate serine proteases in samples butsimultaneously inhibits both the factor C system and factor G systemreactions thereby inhibiting the entire limulus reaction. The presentinvention has been accomplished by further developing these findings.

The present invention provides a reagent for endotoxin assay whichcomprises a limulus amebocyte lysate reagent and aprotinin.

The present invention also provides a kit for endotoxin assay whichcontains a limulus amebocyte lysate reagent and a reagent containingaprotinin.

The present invention further provides a method for endotoxin assayusing a limulus amebocyte lysate reagent, wherein aprotinin is added tothe limulus amebocyte lysate reagent and/or a sample.

Specifically, in the above assay method, aprotinin to be added to thelimulus amebocyte lysate reagent and/or a sample is used in an amounteffective for inhibiting activation of factor G existing in the limulusamebocyte lysate reagent. Further, aprotinin is added to the limulusamebocyte lysate reagent and/or a serin protease-containing sample in aneffective amount for inhibiting the serine protease in the sample.

The present invention further provides a method for assaying endotoxinin a serine protease-containing sample using a limulus amebocyte lysatereagent, wherein the sample is allowed to contact with anaprotinin-immobilized insoluble carrier in advance of the assay.

The present invention further provides an insoluble carrier forpretreating a serine protease-containing sample in advance of endotoxinassay using a limulus amebocyte lysate reagent, on which aprotinin isimmobilized.

Furthermore, the present invention provides a method for inhibitingfactor G activation which comprises adding aprotinin to a limulusamebocyte lysate reagent containing factor G, and a factor G activationinhibitor which comprises aprotinin as an active ingredient capable ofinhibiting activation of factor G existing in the limulus amebocytelysate reagent and a buffer agent for maintaining an optimum pH rangefor a reaction of factor C with endotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mechanism of limulus reaction.

FIG. 2 is a graph showing results of Example 1-2) in which calibrationcurves are obtained by plotting absorbance against endotoxinconcentration as a function of added amounts of aprotinin (- ∘-: notadded, - Δ-: 2.0 mg, - □-: 4.0 mg and - -: 6.0 mg).

DETAILED DESCRIPTION OF THE INVENTION

Aprotinin, also called basic pancreatic trypsin inhibitor, is a basicpolypeptide consisting of 58 amino acid residues and having anisoelectric point of 10.5 extracted from bovine lung, pancreas orparotid gland, which inhibits various intracellular proteases such askallikrein, plasmin, trypsin, chymotrypsin and the like. As apharmaceutical drug, aprotinin is manufactured by Bayer AG under a tradename of TRASYLOL™.

Examples of the limulus amebocyte lysate reagent (hereinafter simplyreferred to as "lysate reagent") used in the present invention includeamebocyte extracts prepared from hemolymph of horseshoe crab, such asLimulus polyphemus, Tachypleus tridentatus, Tachypleus. gigas,Carcinoscorpius rotundicauda and the like in the usual way (cf. J.Blochem., 80, 1011-1021 (1976)). To these extracts may be added adivalent metal salt which is effective for the activation of factor C,such as hydrohalide (e.g., chloride), sulfate or the like of an alkalineearth metal (e.g, magnesium, calcium, strontium or the like), asubstrate for the clotting enzyme, such as the above-mentioned syntheticsubstrate, Boc-Leu-Gly-Arg-pNA, and a pH adjusting agent, such asTris-HCl or the like buffer solution. A commercially available lysatereagent can also be used. The lysate reagent may be in any form such asliquid, powder, solid or the like.

According to the present invention, the lysate reagent containing factorG is preferably used, but those from which factor G has been eliminatedor in which factor G was inactivated with an inhibitor other thanaprotinin may also be used.

The above-mentioned first object of the present invention can beattained by the endotoxin assay which comprises (A) using a reagentprepared by adding aprotinin to a lysate reagent to inactivate factor Gsystem reaction-related components (hereinafter referred to as"aprotinin-containing lysate reagent"), (B) adding aprotinin to a sampleand assaying the sample using a commonly used lysate reagent so thatactivation of factor G system reaction-related components in the lysatereagent can be inhibited, or a combined method of (A) and (B) in whichaprotinin is added to both the lysate reagent and a sample.

In this instance, though the amount of aprotinin necessary forcompletely inhibiting the factor G system reaction depends on the typeof the lysate reagent, one skilled in the art would easily determine theappropriate amount by the following preliminary experiment.

With cooling on an ice bath, aprotinin (endotoxin-free) is added to apredetermined amount of the lysate reagent in a varied amount,endotoxin-free β-glucan is added thereto in such an amount that it canfully activate the lysate reagent under usual measurement conditions andthen the reaction is carried out under the usually used conditions.Under these conditions, the amount of aprotinin which completely inhibitactivation of the lysate reagent by β-glucan is determined.

Based on the thus determined amount of aprotinin, a suitable amount ofaprotinin where the factor C system reaction proceeds corresponding tothe concentration of endotoxin in a sample is determined. The amount ofaprotinin to be used is approximately from 5 mg to 500 mg per 1 ml ofthe lysate reagent.

The above-mentioned second object of the present invention can beattained by the endotoxin assay which comprises (C) allowing aprotininto react with serine protease in a sample and subsequently allowing theresulting reaction mixture to react with the aprotinin-containing lysatereagent, (D) adding aprotinin to a sample in such an amount thatactivity of factor G in the lysate reagent can be inhibited, andallowing the sample to react with a lysate reagent, (E) adding aprotininin advance to the lysate reagent in such an amount that factor G in thelysate reagent can be inhibited as well as serine protease activity in asample or (F) allowing a serine protease-containing sample to contact inadvance with an aprotinin-immobilized insoluble carrier, therebyremoving or inactivating the serine protease and subsequently assayingthe sample thus treated using the lysate reagent.

It should be noted however that the method (E) can be effected whenaprotinin is used within such a range that it does not inhibit thefactor C activity.

Also, in the case of the methods (C) to (E), Et assay can be made moreaccurately and simply using the Et-specific lysate reagent sinceaprotinin is used in an amount required for inhibiting only serineprotease activity and thus the factor C system reaction can becontrolled more easily. In that case, it is desirable to eliminate theamount of aprotinin required for inhibiting factor G from theabove-mentioned range of aprotinin amount. The amount may not be reducedunless the provided amount is effective for endotoxin assay.

In the case of the methods (C) to (E), the sample may be a serineprotease preparation (in other words, the case where the presence of Etin the serine protease preparation is determined) or the sample may becontaminated with a serine protease. In that case, the amount ofaprotinin can be determined in the following manner.

In a synthetic substrate method, aprotinin having a varied concentrationand a sample are added to the limulus reagent for synthetic substratemethod use in which its lysate component is replaced by distilled water,the resulting mixture is incubated at 37° C. to effect the usual limulusreaction and then the amount of aprotinin showing the same value as thatof the blank test is determined, which can be used for assaying thesample.

In a gelation method or a turbidity method, aprotinin having a variedconcentration and a sample are added to the lysate reagent in whichfactor C and factor G are removed or inactivated and coagulogen iscontained, the resulting mixture is incubated at 37° C. to effect theusual limulus reaction and then the amount of aprotinin showing the samevalue as that of the blank test is determined. The Et assay can becarried out by adding the thus determined amount of aprotinin to thesample.

In this instance, the above-mentioned amount of aprotinin is a desiredvalue and therefore can be increased slightly within such a range thatit does not affect the endotoxin assay.

Though it depends on the type and amount of serine protease contained inthe sample, aprotinin may be used in an amount sufficient for completelyinhibiting the serine protease contained in the sample and does notsubstantially interfere with the factor C system reaction. Specifically,aprotinin can be added in general in an amount of approximately from 1to 2,000 moles per mole of the protease, or approximately from 300 μg to10 mg in a practical manner.

In consequence, when a sample to be tested contains a serine proteaseand the lysate reagent contains factor G, the amount of aprotinin is thesum of those required for inhibiting both factor G and serine protease.

In the case of the method (F), the amount of aprotinin to be immobilizedto an insoluble carrier can be determined based on the amount of aserine protease contained in each sample and the like factors, butgenerally within the range of from 2 to 100 moles per mole of the serineprotease. The insoluble carrier is not particularly limited providedthat it does not contain Et and can chemically immobilize aprotininwithout reducing desired activity. Illustrative examples of suchcarriers include polyamide compounds, cellulose compounds, agarosecompounds, polyacrylamide compounds, dextran compounds, vinyl polymercompounds (porous copolymers with glycidyl methacrylate) and the like.Immobilization of aprotinin may be effected by usually used means suchas a method in which formyl groups are introduced into cellulose gel andaprotinin is allowed to bind to the thus treated gel in the presence ofNaCNBH₃, as well as a diazotization method, a CNBr method, an acid azidemethod and the like.

The insoluble carrier can be used in any form. It may be molded to theform of the bead, tip, tube, film and the like. Examples of theinsoluble carrier include any commercially available carrier foraffinity chromatography such as an agarose gel carrier activated by aCNBr method (e.g., Sepharose available from Pharmacia, etc.) and acellulose carrier having an active group such as formyl groups orcarboxyl groups (e.g., Formyl-Cellulofine, Carboxyl-Cellulofine, bothavailable from Seikagaku Corporation, etc.).

In the method (F), a serine protease-containing sample is allowed tocontact with an aprotinin-immobilized insoluble carrier, and theresulting unabsorbed solution is used in the assay. During thistreatment, it is necessary to take special precautions to preventcontamination by Et. According to the methods (C) to (E), Et can besimply and rapidly assayed with minimum danger of contamination of thesample with Et.

The endotoxin assay methods, such as methods (A) to (E), excludingmethod (F), can be carried out in the presence of aprotinin, forexample, by the following methods (i) to (ix).

(i) A method in which aprotinin is added to amebocyte followed byextraction to serve as an aprotinin-containing lysate reagent for the Etassay.

(ii) A method in which aprotinin is added to the extracted lysate toserve as an aprotinin-containing lysate reagent for the Et assay.

(iii) A method in which a lyophilized lysate reagent preparation isdissolved in an aprotinin-containing solution to serve as anaprotinin-containing lysate reagent for the Et assay.

(iv) A method in which aprotinin is added to a lyophilized lysatereagent preparation dissolved in an appropriate solution to serve as anaprotinin-containing lysate reagent for the Et assay.

(v) A method in which a lyophilized preparation, which is prepared byadding aprotinin to amebocyte followed by extraction and lyophilizationor adding a necessary amount of aprotinin to a lysate reagent followedby lyophilization, is dissolved in an appropriate solution to serve asan aprotinin-containing lysate reagent for the Et assay.

(vi) A method in which a lyophilized preparation containing a lysatereagent and a synthetic substrate is dissolved in anaprotinin-containing solution or aprotinin is added to a solutionprepared by dissolving the lyophilized preparation in an appropriatesolution to serve as an aprotinin-containing lysate reagent for the Etassay.

(vii) A method in which a reagent, which is prepared by adding anecessary amount of aprotinin to a mixed solution of a lysate reagentand a synthetic substrate followed by lyophilization, is dissolved in anappropriate solution to serve as an aprotinin-containing lysate reagentfor the Et assay.

(viii) A method in which a necessary amount of aprotinin is added to asample to be tested.

(ix) A method in which a sample to be tested is added to a lysatereagent, followed by immediately adding aprotinin thereto.

In this instance, the method (viii) may be used alone or together withthe methods (i) to (vii).

The solution to be used for dissolving lyophilized preparation in theabove methods (iv), (v), (vi) and (vii) is an appropriate buffer capableof stably maintaining components in the lysate reagent involved infactor C system reaction and maintaining an optimum pH range (pH 7.0 to8.5) for a reaction of factor C with endotoxin. Examples of the solutioninclude water and a buffer solution capable of maintaining the above pHrange which contains a buffer agent such asTris(hydroxymethyl)aminomethane,Tris(hydroxymethyl)-aminomethanemaleate,1,4-piperazinediethanesulfonate, morpholinopropanesulfonate,N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate, triethanolamine,imidazole and Tris(hydroxymethyl)imidazole. The aprotinin-containingsolution used in the above methods (iii) and (vi) is prepared by addinga necessary amount of aprotinin to the above-described solution.

As described above, aprotinin may be used in any optional manner in theEt assay method of the present invention, provided that the factor Csystem reaction in the lysate reagent can function normally or within areasonably functionable range and that quantitative or qualitative assayof Et can be made.

The endotoxin assay using the reagent of the present invention may beeffected in the usual way by determining the activity of clotting enzymeformed by its activation through the cascade reaction shown in FIG. 1.

For the measurement of amidase activity of the clotting enzyme, theabove-mentioned synthetic peptide substrate having a chromogenic residuemay be used, as well as its analogous synthetic peptide substrate whichhas the same peptide sequence but the carboxyl group of its C-terminalamino acid is substituted, in place of the above-mentioned chromogenicresidue, with a known fluorescence residue, a luminescence residue,ammonia or the like via an amide bond. The amidase activity isdetermined by measuring the reaction product formed from the syntheticsubstrate by the action of the clotting enzyme. For example, theabove-described synthetic substrate is allowed to coexist with areaction system which contains the reagent of the present invention andendotoxin, and a dye, a fluorescent substance or ammonia formed by thereaction (cascade reaction and, if necessary, a conversion reaction ofthe reaction product into other dyes) is detected respectively by aspectrophotometer, a fluorophotometer, a chemi-luminescent detector, anammonia detection electrode (JP-A-62-148860; the term "JP-A" as usedherein means an "unexamined published Japanese patent application) orthe like.

Determination of protease activity of the clotting enzyme may beeffected by a method in which the clotting enzyme is allowed to reactwith coagulogen (substrate) which is contained in the reagent of thepresent invention or added separately, and the resulting gelation ofcoagulin is measured, for example, by an appropriate instrument such asa turbidity measuring instrument, a viscosity measuring instrument orthe like or by the naked eye judgement.

In the practice of the assay of the present invention, it is necessaryto use a divalent metal salt effective for the activation of theabove-mentioned cascade reaction system. Examples of such divalent metalsalts include hydrohalides (chloride) and sulfate of alkaline earthmetals such as magnesium, calcium, strontium and the like.

Though these metal salts can be added independently upon the limulusreaction, it is desirable to add the divalent metal salt to the lysatereagent and drying the mixture to solid under a non-heating conditionsuch as lyophilization. The reagent for use in the measurement ofamidase activity may preferably be coexisted with the above-mentionedsynthetic peptide substrate in addition to the divalent metal salt,which may be further dried.

The endotoxin assay according to the present invention can be carriedout more easily and rapidly by using a kit comprising theabove-described reagents. The kit of the present invention comprises thelysate reagent and the aprotinin-containing reagent. Theaprotinin-containing reagent may further comprise a buffer agent asdescribed above. Specific example of the kit includes: (1) a lyophilizedlysate reagent and a solution for dissolving it in; (2) lyophilizedaprotinin and a solution for dissolving it in; (3) lyophilized standardendotoxin and a solution for dissolving it in; and (4) reagents forsynthetic chromogenic substrate method including distilled water forblank test.

Samples to be tested for the Et assay is not particularly limited, andany sample required for the quantitative measurement of Et orconfirmation of its presence can be used. Examples of such samplesinclude biological samples, drugs, water for medical use and the like.As described above, the present invention is especially useful for theassay of serine protease-containing samples.

According to the present invention, types of the serine proteasescontained in samples to be tested are not particularly limited, providedthat their activities are inhibited by aprotinin. Examples of suchserine proteases include kallikrein, plasmin, trypsin, chymotrypsin,thrombin and the like.

The following examples are provided to further illustrate the presentinvention, but are not to be understood to limit the scope of theinvention.

EXAMPLE 1

Et assay using a reagent prepared by adding aprotinin to a lysatereagent

1) A 1.0 liter portion of hemolymph of T. tridentatus was centrifuged at1,500 rpm at 4° C. for 10 minutes, and about 21 g of the precipitate(amebocytes) thus obtained was mixed with 210 ml of 0.02M Tris-HClbuffer (pH 8.0) and homogenized uniformly in a homogenizer (Polytron RPT10, the trade name of the product manufactured by Kinematica) toeffect extraction. The resulting homogenate was subjected tocentrifugation at 10,000×G for 30 minutes under ice-cooling to obtain190 ml of supernatant (lysate reagent).

To 0.04 ml of the thus obtained lysate reagent were added 0.04 ml of0.5M Tris-HCl/0.4M magnesium sulfate buffer (pH 8.0) containing 0.5,1.0, 2.0, 4.0 or 6.0 mg of aprotinin and 0.02 ml of 4.0 mMBoc-Leu-Gly-Arg-pNA, thereby obtaining an aprotinin-containing lysatereagent (invention). Separately, to 0.04 ml of the lysate reagent wereadded 0.04 ml of 0.5M Tris-HCl/0.4M magnesium sulfate buffer (pH 8.0)containing no aprotinin and 0.02 ml of 4.0 mM Boc-Leu-Gly-Arg-pNA. Toeach of these was added 0.1 ml of distilled water (blank, hereinafterreferred to as "DW") or β-glucan (500 ng/ml, prepared by the methoddescribed below) as a sample to be tested. After allowing each of theresulting mixtures to react at 37° C. for 30 minutes, the thus formedp-nitroaniline was subjected to diazo-coupling by adding to the reactionmixture 0.5 ml each of 0.04% sodium nitrite (0.48M hydrochloric acidsolution), 0.3% ammonium sulfamate and 0.07%N-1-naphthylethylenediaminedihydrochloride in that order. Thereafter,absorbances were measured at 545 nm and the difference between those ofeach sample and blank was regarded as the reactivity. The results areshown in Table 1 (the difference is expressed by "Δ" in the table andalso in other tables and figures).

                  TABLE 1                                                         ______________________________________                                        Amount of                                                                     aprotinin (mg)                                                                            Reactivity (ΔA.sub.545 nm/30 min)                           ______________________________________                                        0           >1.5                                                              0.5         0.437                                                             1.0         0.153                                                             2.0         0.000                                                             4.0         0.000                                                             6.0         0.000                                                             ______________________________________                                    

As is evident from Table 1, activation of factor G in the lysate reagentcan be inhibited completely when 2.0 mg or more of aprotinin is added to0.04 ml of the lysate reagent.

2) To 0.04 ml of the lysate reagent prepared in the above 1) were added0.04 ml of 0.5M Tris-HCl/0.4M magnesium sulfate buffer (pH 8.0)containing 2.0, 4.0 or 6.0 mg of aprotinin, which completely inhibitedfactor G system reaction in the lysate reagent, and 0.02 ml of 4.0 mMBoc-Leu-Gly-Arg-pNA to obtain an aprotinin-containing lysate reagent.Separately, to 0.04 ml of the lysate reagent were added 0.04 ml of 0.5MTris-HCl/0.4M magnesium sulfate buffer (pH 8.0) containing no aprotininand 0.02 ml of 4.0 mM Boc-Leu-Gly-Arg-pNA. To each of these was added0.1 ml of DW (blank) or Et derived from Escherichia coli 0111:B4(Westphal type, available from Sigma; 6.25, 12.5, 25.0 or 50.0 pg/ml) asa sample. Thereafter, the reaction was effected in the same manner asdescribed in the above 1) to prepare calibration curves of Et. Theresults are shown in FIG. 2.

It can be found from FIG. 2 that the reactivity with Et decreases as theamount of added aprotinin increases. By the use of these calibrationcurves, the amount of aprotinin which gives activity of the factor Csystem reaction in the lysate corresponding to the Et concentration ineach sample can be selected.

Further, it is evident that Et assay can be effected using a limulusreagent which is prepared by adding aprotinin to a usual lysate reagentwithout influence of β-glucan.

Preparation of β-glucan

The method disclosed in WO90/02951 was followed. One gram of curdlan(available from Wako Pure Chemical Industries) was suspended in about100 ml of a 5 mM NaOH aqueous solution and sonicated by Sonicator™(Model 5202 PZT, manufactured by Ohtake Seisakusho, Tokyo) at 20 KHz and80 W for 12 minutes under ice-cooling to degrade curdlan. The resultingsolution was adjusted to give a final NaOH concentration of 0.3M with a5M NaOH aqueous solution and subjected to a gel permeationchromatography (GPC columns, two TSK gel G3000PW_(XL) and oneG2500PW_(XL) ; mobile phase, 0.3M NaOH aqueous solution; flow rate, 0.5ml/min). The resulting fractions were pooled and again subjected to thechromatography to collect a fraction of a molecular weight of 216,000,thereby obtaining a GPC-fractionation purified β-glucan preparation.

The thus obtained β-glucan preparation was also used in the followingexamples.

EXAMPLE 2

Et assay using a reagent prepared by adding aprotinin to amebocytefollowed by extraction

A 1.0 liter portion of hemolymph of T. tridentatus was centrifuged at1,500 rpm at 4° C. for 10 minutes and about 21 g of the resultingprecipitate (amebocytes) was mixed with 210 ml of 0.02M Tris-HCl buffer(pH 8.0) containing 12 g of aprotinin and uniformly homogenized inPolytron R PT10 to effect extraction. The resulting homogenate wassubjected to centrifugation at 10,000×G for 30 minutes while cooling toobtain 190 ml of a supernatant (aprotinin-containing lysate reagent).

To 0.04 ml of each of the thus obtained aprotinin-containing lysatereagent (invention) and a lysate reagent prepared in the same manner butwithout adding aprotinin (comparison) were added 0.01 ml of 2M Tris-HClbuffer (pH 8.0), 0.03 ml of 0.4M magnesium chloride and 0.02 ml of 3.0mM Boc-Leu-Gly-Arg-pNA, followed by the addition of 0.1 ml of DW(blank), Et or β-glucan as a sample. Further, 0.05 ml each of Et andβ-glucan having two-fold concentration were added as another sample.Each of the resulting mixtures was incubated at 37° C. for 30 minutes,the reaction was terminated with 0.4 ml of 0.8M acetic acid and then thethus formed p-nitroaniline was determined by measuring absorbance at 405nm to compare reactivities. The results are shown in Table 2.

From the results, it is evident that Et assay can be effected using anaprotinin-containing lysate reagent prepared by adding aprotinin to alimulus hemolymph amebocyte prior to extraction without influence ofβ-glucan. In other words, it was confirmed that the factor G systemreaction is substantially inhibited without inhibiting the factor Csystem reaction in the assaying system of the present invention.

                  TABLE 2                                                         ______________________________________                                                      Reactivity (ΔA.sub.405 nm/30 min)                         Samples         Invention                                                                              Comparison                                           ______________________________________                                        Et*             0.317    0.344                                                β-Glucan** 0.000    0.183                                                glucanbeta.     0.317    0.526                                                ______________________________________                                         *Et concentration: 3.0 pg/0.1 ml sample                                       **β-glucan concentration: 5.0 pg/0.1 ml sample                      

EXAMPLE 3

Et assay using a reagent prepared by dissolving a lyophilized lysatereagent in an aprotinin-containing solution

One vial portion of "Pyrotel-T" (a lyophilized preparation of a lysatereagent prepared from L. polyphemus for a gelation method use,manufactured by Capecod and available from Seikagaku Corporation) wasdissolved in 5.0 ml of DW in which 270 mg of aprotinin had beendissolved in advance (invention). Another one vial portion of thelyophilized limulus reagent preparation was dissolved in 5.0 ml of DWcontaining no aprotinin (comparison). A 0.1 ml aliquot of thesesolutions was dispensed in 0.1 ml portions into test tubes, followed byaddition of 0.1 ml of DW (blank), Et or β-glucan as a sample. Further,0.05 ml each of Et and β-glucan both having two-fold concentration wereadded as another sample. After gently mixing the reaction mixture, eachtest tube was arranged on an analysis module attached to a nephelometricanalyzer (Toxinometer ET-201, available from Wako Pure ChemicalIndustries) and incubated at 37° C. for 60 minutes to record gelationtime (Tg) and thereby to examine reactivity of the reagent of thepresent invention. The results are shown in Table 3.

From the results, it is evident that Et assay can be effected by addingaprotinin to a commercially available lyophilized lysate reagent(gelation method limulus test reagent) prior to the addition of sampleswithout influence of β-glucan.

                  TABLE 3                                                         ______________________________________                                                      Reactivity (Tg, min)                                            Sample          Invention                                                                              Comparison                                           ______________________________________                                        DW (blank)      >60      >60                                                  Et*             33.5     29.7                                                 β-Glucan** >60      32.3                                                 glucanbeta.     33.3     19.6                                                 ______________________________________                                         *Et concentration: 2.0 pg/0.1 ml sample                                       **β-glucan concentration: 40.0 pg/0.1 ml sample                     

EXAMPLE 4

Et assay using a reagent prepared by dissolving a lyophilizedpreparation containing a lysate reagent and a synthetic substrate in anaprotinin-containing solution

One vial portion of "Toxicolor System LS-200 Set" (a lyophilizedpreparation containing a lysate reagent prepared from T. tridentatus andBoc-Leu-Gly-Arg-pNA for chromogenic synthetic substrate method use,manufactured by and available from Seikagaku Corporation) was dissolvedin 2.8 ml of 0.2M Tris-HCl buffer (pH 8.0) in which 140 mg of aprotininhad been dissolved in advance to obtain a reagent of the presentinvention. Another one vial portion of the lyophilized preparation wasdissolved in 2.8 ml of 0.2M Tris-HCl buffer (pH 8.0) containing noaprotinin to serve as a comparative reagent. To 0.1 ml of each of thesesolutions was added 0.1 ml of DW (blank), Et or β-glucan as a sample tobe tested. Further, 0.05 ml each of Et and β-glucan both having two-foldconcentration were added as another sample. Each of these mixtures wasallowed to react in the same manner as described in Example 1--1) toevaluate reactivity of the reagent of the present invention. The resultsare shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                      Reactivity (ΔA.sub.545 nm/30 min)                         Sample          Invention                                                                              Comparison                                           ______________________________________                                        Et*             0.863    0.891                                                β-Glucan** 0.000    >1.5                                                 glucanbeta.     0.863    >1.5                                                 ______________________________________                                         *Et concentration: 5.0 pg/0.1 ml sample                                       **β-glucan concentration: 50.0 ng/0.1 ml sample                     

From the results, it is evident that Et assay can be effected by addingaprotinin to a commercially available chromogenic synthetic substratemethod limulus test reagent (lyophilized preparation) prior to theaddition of sample without influence of β-glucan.

EXAMPLE 5

Et assay using a reagent prepared by adding aprotinin to amebocytefollowed by extraction, lyophilizing the mixture and dissolving it in asolution

A 2.0 ml portion of an aprotinin-containing lysate reagent prepared byadding aprotinin to amebocyte prior to extraction in the same manner asin Example 2 was mixed with 0.4 ml of 0.4M magnesium chloride followedby lyophilization to obtain a reagent for Et assay of the presentinvention. Also, 2.0 ml of an aprotinin-free lysate reagent prepared inthe same manner as in Example 1--1) was mixed with 0.4 ml of 0.4Mmagnesium chloride followed by lyophilization to obtain a comparativereagent. Each of the thus lyophilized preparations was dissolved in 2.0ml of DW. To 0.1 ml of each of these solutions was added 0.1 ml of DW(blank), Et or β-glucan as a sample. After gentle mixing, the mixturewas statically incubated at 37° C. for 60 minutes and then the presenceof gel formation was judged by the naked eye by slanting the tube at anangle of 180° to evaluate reactivity of the reagent of the presentinvention. The results are shown in Table 5. In the table, "+" means gelformation and "-" means no gel formation.

                  TABLE 5                                                         ______________________________________                                                     Reactivity                                                       Sample         Invention                                                                              Comparison                                            ______________________________________                                        DW (blank)     -        -                                                     Et*            +        +                                                     β-Glucan**                                                                              -        +                                                     ______________________________________                                         *Et concentration: 4.0 pg/0.1 ml sample                                       **β-glucan concentration: 40.0 ng/0.1 ml sample                     

From the results, it is evident that Et assay can be effected bylyophilizing an aprotinin-containing lysate reagent prepared by addingaprotinin to amebocyte prior to extraction without influence ofβ-glucan.

EXAMPLE 6

Et assay using a reagent prepared by dissolving in a solution alyophilized preparation containing a lysate reagent, a syntheticsubstrate and aprotinin

Mixed were 2.0 ml of the aprotinin-free lysate reagent obtained inExample 1--1), 0.9 ml of 3.4 mM chromogenic synthetic substrate(Boc-Leu-Gly-Arg-pNA), 1.0 ml of 0.8M magnesium sulfate and 0.5 ml ofaprotinin aqueous solution (240 mg/ml). The mixture was lyophilized toobtain a reagent for Et assay of the present invention. Also, acomparative reagent was prepared in the same manner except that 0.5 mlof DW was used in place of the aprotinin aqueous solution. Each of thethus obtained lyophilized preparations was dissolved in 5.0 ml of 0.2MTris-HCl buffer (pH 8.0). To 0.1 ml of each of the resulting solutionswas added 0.1 ml of DW (blank), Et or β-glucan as a sample. Further,0.05 ml each of Et and β-glucan both having two-fold concentration wereadded as another sample. Each of these mixtures was allowed to react inthe same manner as described in Example 1--1) to evaluate reactivity ofthe reagent of the present invention. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                      Reactivity (ΔA.sub.545 nm/30 min)                         Sample          Invention                                                                              Comparison                                           ______________________________________                                        Et*             0.669    0.697                                                β-Glucan** 0.000    >1.5                                                 glucanbeta.     0.669    >1.5                                                 ______________________________________                                         *Et concentration: 4.0 pg/0.1 ml sample                                       **β-glucan concentration: 40.0 ng/0.1 ml sample                     

From the results, it is evident that Et assay can be effected by using areagent prepared by lyophilizing a mixture containing a lysate reagent,a synthetic substrate and aprotinin without influence of β-glucan.

EXAMPLE 7

Et assay in which aprotinin is added in advance to samples

DW (blank), Et, β-glucan and a mixture of the same volume of Et andβ-glucan both having two-fold concentration were prepared as samples tobe tested. To 0.05 ml of each of these samples were added 0.05 ml ofaprotinin aqueous solution (100 mg/ml) and then 0.1 ml of "ToxicolorSystem LS-200 Set" which had been dissolved in 2.8 ml of 0.2M Tris-HClbuffer (pH 8.0). Thereafter, each of the thus prepared mixtures wasallowed to react in the same manner as described in Example 1--1). Thesame procedure was repeated except that the same volume of DW was usedin place of the aprotinin aqueous solution in order to compare theirreactivities. The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                   Reactivity (ΔA.sub.545 nm/30 min)                            Sample       Aprotinin added                                                                           Aprotinin not added                                  ______________________________________                                        Et*          0.883       0.928                                                β-Glucan**                                                                            0.000       >1.5                                                 glucanbeta.  0.885       >1.5                                                 ______________________________________                                         *Et concentration: 5.0 pg/0.05 ml sample.                                     **β-glucan concentration: 30.0 ng/0.05 ml sample                    

From the results, it is evident that Et assay can be effected by addingaprotinin in advance to samples without influence of β-glucan.

EXAMPLE 8

Et assay in which a sample is added to a lysate reagent and immediatelythereafter aprotinin is added thereto

One vial portion of "Pyrotel" (a lyophilized preparation of a lysatereagent prepared from L. polyphemus for a gelation method use,manufactured by Capecod and available from Seikagaku Corporation) wasdissolved in 5.0 ml of DW. The thus prepared solution was dispensed intotest tubes in 0.1 ml portions under ice-cooling and 0.05 ml of DW(blank), Et or β-glucan was added thereto as a sample. Immediatelythereafter, 0.05 ml of an aprotinin aqueous solution (110 mg/ml) wasadded to the respective test tubes. After gentle mixing, each of thesemixtures was incubated statically at 37° C. for 60 minutes to judge thepresence of gel formation in the same manner as described in Example 5.The same procedure was repeated except that the same volume of DW wasused in place of the aprotinin aqueous solution in order to comparetheir reactivities. The results are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                  Reactivity                                                          Sample      Aprotinin added                                                                           Aprotinin not added                                   ______________________________________                                        DW (blank)  -           -                                                     Et*         +           +                                                     β-Glucan**                                                                           -           +                                                     ______________________________________                                         *Et concentration: 2.0 pg/0.05 ml sample                                      **β-glucan concentration: 20.0 ng/0.05 ml sample                    

From the results, it is evident that Et assay can be effected by addingsamples to a lysate reagent and immediately thereafter adding aprotininthereto without influence of β-glucan.

EXAMPLE 9

Et assay in which aprotinin is added to serine protease-containingsamples

A 0.01 ml portion of DW or Et solution (400 pg/ml) was added to 0.01 mlof a 100 μg/ml solution of trypsin (derived from bovine pancreas,manufactured by Miles Laboratories and available from SeikagakuCorporation) or DW, and 0.08 ml of an aprotinin aqueous solution (5.0mg/ml) was added thereto followed by mixing. Each of the thus preparedmixtures was further mixed with 0.1 ml of a limulus test reagent forEt-specific chromogenic synthetic substrate method use (Endospecy,available from Seikagaku Corporation) and subjected to the same reactionas described in Example 1--1) to determine recovery of Et added to thetrypsin sample. The same procedure was repeated except that 0.08 ml ofDW was used in place of the aprotinin aqueous solution. The results areshown in Table 9.

                  TABLE 9                                                         ______________________________________                                               Addition of        Et not                                                     aprotinin Et added added         Recovery                              Sample to samples                                                                              (A.sub.545 nm)                                                                         (A.sub.545 nm)                                                                       ΔA.sub.545 nm                                                                  of Et (%)                             ______________________________________                                        DW     yes       0.729    0.070  0.659  100                                   Trypsin                                                                              yes       0.743    0.095  0.648  98.3                                  DW     no        0.701    0.031  0.670  100                                   Trypsin                                                                              no        >1.5     >1.5   --     --                                    ______________________________________                                    

As is evident from the results shown in Table 9, Et in atrypsin-containing sample can be assayed accurately by a limulus test byadding aprotinin to the trypsin-containing sample in advance.

EXAMPLE 10

Et assay of serine protease-containing samples in which aprotinin isadded to a lysate reagent

A 0.08 ml portion of an aprotinin aqueous solution (8 mg/ml) was addedto 0.1 ml of Toxicolor System LS-200 Set followed by mixing. To this wasadded 0.02 ml of a solution prepared by mixing a 10 units/ml solution ofthrombin (derived from bovine serum, available from Sigma) or DW withthe same volume of Et solution (400 pg/ml). Each of the thus preparedmixtures was subjected to the same reaction as described in Example1--1) to determine recovery of Et added to the thrombin sample. The sameprocedure was repeated except that 0.08 ml of DW was used in place ofthe aprotinin aqueous solution. The results are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                              Addition of         Et not                                                    aprotinin to                                                                             Et added added         Recovery                              Sample                                                                              lysate reagent                                                                           (A.sub.545 nm)                                                                         (A.sub.545 nm)                                                                       ΔA.sub.545 nm                                                                  of Et (%)                             ______________________________________                                        DW    yes        0.718    0.068  0.650  100                                   Throm-                                                                              yes        0.764    0.116  0.648  99.7                                  bin                                                                           DW    no         0.693    0.028  0.665  100                                   Throm-                                                                              no         >1.5     >1.5   --     --                                    bin                                                                           ______________________________________                                    

As is evident from the results shown in Table 10, Et in athrombin-containing sample can be assayed accurately by adding aprotininto a limulus test reagent.

EXAMPLE 11

Et assay in which serine protease-containing samples are pretreated withan aprotinin-immolized insoluble carrier prior to limulus test

A 10 ml portion of a 200 μg/ml solution of trypsin (derived from swinepancreas, manufactured by Sigma) was applied to a column (1.2×4.0 cm)packed with Et-free aprotinin-immobilized cellulofine (prepared by themethod described below) which had been equilibrated with 0.1M Tris-HClbuffer (pH 8.0) containing 0.15M NaCl . The column was then washed with0.1M Tris-HCl buffer (pH 8.0) containing 0.15M NaCl, and fractionspassed through the column were pooled. To 0.1 ml of the pooled fractionor 0.1 ml of DW was added 0.01 ml of an Et solution (400 pg/ml) andsubsequently 0.1 ml of Endospecy. The resulting mixture was allowed toreact in the same manner as in Example 1--1) to determine recovery of Etin the fraction. The same procedure was repeated except that 0.01 ml ofDW or an Et solution and subsequently 0.1 ml of Endospecy were added to0.1 ml of untreated trypsin solution. The results are shown in Table 11.

                  TABLE 11                                                        ______________________________________                                                 Et        Et not                                                              added     added             Recovery                                 Sample   (A.sub.545 nm)                                                                          (A.sub.545 nm)                                                                          ΔA.sub.545 nm                                                                   of Et (%)                                ______________________________________                                        DW       0.709     0.032     0.677   100                                      Passed-through                                                                         0.710     0.034     0.676   99.9                                     fraction                                                                      Untreated                                                                              >1.5      >1.5      --      --                                       trypsin                                                                       ______________________________________                                    

As is evident from the results shown in Table 11, Et in atrypsin-containing sample can be assayed accurately by limulus test whentrypsin is allowed to contact in advance with an aprotinin-immobilizedinsoluble carrier.

Preparation of aprotinin-immobilized Cellulofine

A 10 g portion of Formyl-Cellulofine (manufactured by Chisso andavailable from Seikagaku Corporation) was washed thoroughly with 0.1Mphosphate-Na buffer (pH 7.1), suspended in 20 ml of an aprotininsolution (20 mg/ml in 0.1M phosphate-Na buffer, pH 7.1). Fifty mg ofNaCNBH₃ was added to the suspension. After gently stirring at roomtemperature for 8 hours, the suspension was washed with 0.2M Tris-HClbuffer (pH 7.0) and filtered. Five ml of the above buffer containing 10mg of NaCNBH₃ was added to the resulting residue followed by shaking atroom temperature for 3 hours. Thereafter, the thus obtained suspensionwas washed thoroughly with 0.1M Tris-HCl buffer (pH 8.0) containing0.15M NaCl.

EXAMPLE 12

The procedure of Example 2 was repeated except for using as a sample 0.1ml of platelet rich plasma obtained from a patient who was suspected tosuffer from complicated sepsis which was treated with perchloric acidaccording to the method described in U.S. Pat. No. 4,495,294 and thenneutralized. The sample was also cultured according to the usual methodto detect microbial infection. As a result, a number of E. coli colonieswere detected in conformity with the quantitative reactivity of Et.

Also, effectiveness of the reagent or method of the present inventionwas confirmed when the same sample was tested in accordance with theprocedures of Examples 3 to 8.

Thus, the reagent for Et assay can be prepared by such a simple andeconomical manner that a lysate reagent is combined with aprotinin. Itis especially useful for assaying clinical samples from patients who aresuspected to suffer from infectious diseases or sepsis, which are notproved to contain endotoxin, and it can accurately judge true Gramnegative bacterial infection (endotoxemia). In addition, the presentinvention is also advantageous in that endotoxin contained in variousserine protease-containing samples can be assayed easily and accurately,whereas such samples cannot be assayed by the conventional limulus testor need to be subjected to complicated pretreatment because serineproteases strongly interfere the limulus reaction.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for specifically assaying for endotoxinin a sample comprising the steps of:(A) adding aprotinin to a horseshoecrab amebocyte lysate reagent, (B) adding the resulting lysate reagentof step (A) to the sample, and (C) assaying for endotoxin in theresulting mixture of Step (B), wherein said aprotinin is added to thelysate reagent in Step (A) in an amount sufficient to completely inhibitactivation of factor G present in the horseshoe crab amebocyte lysatereagent.
 2. The method according to claim 1, wherein assaying forendotoxin in Step (C) is carried out by determining the activity ofclotting enzyme.
 3. A method for specifically assaying for endotoxin ina sample comprising the steps of:(A) adding aprotinin to the sample, (B)adding a horseshoe crab amebocyte lysate reagent to the resulting sampleof Step (A), and (C) assaying for endotoxin in the resulting mixture ofStep (B), wherein said aprotinin is added to the sample in Step (A) inan amount sufficient to completely inhibit activation of factor Gpresent in the horseshoe crab amebocyte lysate reagent.
 4. The methodaccording to claim 3, wherein assaying for endotoxin in Step (C) iscarried out by determining the activity of clotting enzyme.
 5. A methodfor specifically assaying for endotoxin in a sample comprising the stepsof:(A) adding aprotinin to a horseshoe crab amebocyte lysate reagent,(B) adding aprotinin to the sample, (C) combining the resulting lysatereagent of step (A) and the resulting sample of step (B) to form amixture, and (D) assaying for endotoxin in the mixture of step (C),wherein said aprotinin is added to the lysate reagent of Step (A) and tothe sample of Step (B) in an amount sufficient to completely inhibitactivation of factor G present in the horseshoe crab amebocyte lysatereagent.
 6. The method according to claim 5, wherein assaying forendotoxin in Step (D) is carried out by determining the activity ofclotting enzyme.
 7. A method for inhibiting activation of factor Gpresent in a horseshoe crab amebocyte lysate reagent, comprising thestep of adding aprotinin to said lysate reagent, wherein said aprotininis added in an amount sufficient to completely inhibit activation offactor G in said reagent.